Project #2. Discovery and mechanistic understanding of functionally-selective D2 dopamine receptor ligands (Richard Mailman, Project PI) A. SPECIFIC AIMS The idea that some ligands can selectively activate only some of the signaling pathways mediated by a single receptor isoform ("functional selectivity") is a novel concept that has markedly changed classic concepts of receptor pharmacology. During the past few years, there has been overwhelming support for this concept from more than a dozen receptor systems. We, and almost all of the other labs who have worked in this arena, have recognized that it has profound implications for drug discovery. As discussed in the overall introduction to this application, the functional selectivity hypothesis predicts that functionally selective drugs (i.e., those that differentially activate (or inhibition) signaling pathways linked to a single receptor) will be differentiated not only by their behavior in vitro, but that this will lead to different behavioral and physiological effects seen both preclinically and clinically. This project will focus on the dopamine D2L receptor, and utilize a battery of signaling assays which are independent (or largely independent) of each other. These assays allow the differential classification of functionally selective properties, and allow the rational selection of agents with novel properties. These assays, as well as new assays in development, will allow for the discovery and rational classification of novel functionally selective dopamine agonists. Novel leads identified from in vitro screening campaigns will be profiled computationally and physically for off-target actions and suitably selective compounds will subsequently be evaluated in a variety of behavioral assays that will offer clues predictive of potential therapeutic action. We recognize that the current state-of-the-art is not adequately advanced such that it is possible to predict from a novel pattern of signaling activity seen in vitro whether the candidate ligand might have clinical superiority in discrete domains of psychopathology. In this regard, however, the examination of interesting candidate compounds both in traditional behavioral models and in mice with specific genetic alterations will provide a powerful way to select compounds that might become drug candidates. As an example, one inherent hypothesis is that functionally selective drugs can be discovered that cause significant facilitation of dopaminergic functions that modulate working memory, while still effectively attenuating dopaminergic function that decreases positive symptoms of schizophrenia. The foundation of research from the past two decades makes us believe that we can confirm this optimistic hypothesis. We shall pursue this via the following aims that are heavily integrated with Projects 1 and 3, and will make use of both scientific cores. 1. AIM 1. CHARACTERIZE EXISTING AND DISCOVERY COMPOUNDS FOR FUNCTIONALLY SELECTIVE PROPERTIES IN NON-NEURONAL D2L DOPAMINE RECEPTOR SYSTEMS Hypothesis: A battery of signaling assays that are independent or largely independent of each other will differentiate drugs with functionally selective properties, and allow selection of agents with novel properties. Corollary: The pattern of signaling effects will differentiate the ligands one from the other and be predictive of preclinical behavioral and clinical differences, although not necessary clinical superiority. The goal is to discover novel functionally selective D2 signalers that can provide new lead molecules, and/or ligands that may be useful research tools. The broad array of existing D2L functional assays are expressed in non-neuronal cell lines (CHO and HEK). Although it would be ideal to know the exact mechanisms about how the D2L receptor signals in situ in primate tissue, this knowledge does not currently exist (although it will be explored in Project 1. On the other hand, the current battery can discriminate functionally selective D2 ligands that have unique behavioral properties in animals and humans, providing reasonable evidence of validity. We shall focus on relatively small subsets of rationally selected compounds from our Wyeth coinvestigators, from compounds from our recent research, from promising targets we have found in the public domain, and from selected structures available in commercial libraries predicted from our computational approaches. Reference compounds will be dopamine and two "typical" D2L agonists (quinpirole and RNPA), as well as known functionally selective compounds with different patterns of activity (e.g., aripiprazole and S-3- PPP). The compounds will be analyzed in four receptor-mediated functional assays (inhibition of adenylate cyclase, stimulation of MAPK kinase, arachidonic acid release, and stimulation of GTPase binding). Emax and ED50 values will be determined, and functionally selective ligands for detailed study by this Project and Projects 2 and 3 will be those in which there is greater than a 30% difference intrinsic activity in one or more functional endpoints versus the others, or when the ED50 changes by more than ten-fold relative to the reference compounds. Finally, these assays are currently semi-automated, and provide adequate throughput for the focused types of studies we are making now. In parallel, we shall make efforts to increase their throughput rate (without decreasing either accuracy or precision), such that they can be applied to higher throughput analysis for the D2i_ and other receptors. 2. AIM 2: DETERMINATION OF THE STRUCTURAL BASIS FOR ACTIVATION AND FUNCTIONAL SELECTIVITY OF D2L RECEPTORS. Our hypothesis that functional selectivity results from the ligand-unique sets of induced conformations (rather than selection of discrete active states) makes it important to understand some of the structural determinants that are involved. Thus, we hypothesize that functional selectivity can result either from conformational perturbation of the D2|_ receptor done by ligands "sterically" (close to the residues that bind dopamine) and/or "allosterically" (involving both the steric sites and aspects of the receptor not normally engaged by dopamine). This aim will elucidate some of the subtle structural interactions that differentiate how non-selective and functionally selective ligands affect the D2i_ receptor, and how this results in selective activation of specific effector pathways. Computationally-predicted mutations of the D2|_ receptor will be made, and ligand analysis conducted using rigid or semi-rigid compounds that minimize possible clocking poses, as well as functionally selective ligands emerging from Aim 1. 3. AIM 3. DEVELOP NEURONALLY-RELEVANT CELL SPECIFIC ASSAY SYSTEMS Project #1 seeks to develop physiologically relevant ex vivo models for screening for functionally selective drugs. In parallel, we wish to find immortal cell lines that functionally mirror dopamine neurons. Prior work with the MN9D cell line has shown that these cells provide excellent predictability on which functionally selective drugs produce novel behavioral characteristics in vivo. Unfortunately, we have found this like to be unstable, and seek a line that can replace it. We have identified two excellent candidates for our purposes (N27 and CAD), and we shall first characterize these lines for appropriate phenotype, and for their stability when molecularly-manipulated.